Isolation and structure-function characterization of a signaling-active rhodopsin-G protein complex

Abstract

The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (G_T). This results in the dissociation of G_T into its component α_T-GTP and β₁γ₁ subunit complex. Structural information for the Rho*-G_T complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (G_T*) comprising a Gα_T/Gα_i1 chimera (α_T*) and β₁γ₁ The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to Gα_T* is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one G_T*. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the β₂-adrenergic receptor-G_S complex, including a flexible α_T* helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex.

Keywords: 7-helix receptor; EM; G protein-coupled receptor (GPCR); G-protein; SAXS; photo-transduction; rhodopsin; signal transduction; structural biology; transducin.

Figure 1.

Rho*-catalyzed nucleotide exchange activity of α_T* in various detergents. A, tryptophan fluorescence emission profiles of Rho*-catalyzed nucleotide exchange in maltoside detergents at a concentration of 2× CMC (OM, octyl maltoside; NM, nonyl maltoside; DM, decyl maltoside; UM, undecyl maltoside; DDM, dodecyl maltoside). Inset, rate constants obtained from single exponential fits of the data (n = 3). The respective rate constants are as follows: OM, 0.96 ± 0.13 min⁻¹; NM, 2.81 ± 0.21 min⁻¹; DM, 3.83 ± 0.01 min⁻¹; UM, 2.60 ± 0.67 min⁻¹; DDM, 1.52 ± 0.17 min⁻¹. B, tryptophan fluorescence emission profiles of Rho*-catalyzed nucleotide exchange in maltose neopentyl glycol detergents at a concentration of 2× CMC (OMNG, octyl maltose neopentyl glycol; DMNG, decyl maltose neopentyl glycol; LMNG, lauryl maltose neopentyl glycol). Inset, rate constants obtained from single exponential fits of the data (n = 3). The respective rate constants as follows: OMNG, 1.83 ± 0.52 min⁻¹; DMNG, 3.12 ± 0.56 min⁻¹; LMNG, 2.64 ± 0.68 min⁻¹. C, tryptophan fluorescence emission profiles of Rho*-catalyzed nucleotide exchange in DDM at various detergent concentrations (CMC = 0.0087% w/v). D, tryptophan fluorescence emission profiles of Rho*-catalyzed nucleotide exchange in LMNG at various detergent concentrations (CMC = 0.001% w/v).

Figure 2.

Purification of the Rho*–G_T* complex. A, purification scheme (see “Experimental procedures”). B, SDS-polyacrylamide gel of the purification process. (This is representative of more than 20 repetitions of the purification process.)

Figure 3.

Stoichiometry determination and SEC profiles of the purified Rho*–GT* complex. A, SEC profiles of the purified Rho*–GT* complex (red) and its dissociation upon the addition of GTPγS (green). (This is representative of more than 10 similar sets of SEC profiles.) B, UV-visible spectrum of purified complex. (This is representative of more than 20 similar spectra of the purified complex.) C, ratio of Rho* to G_T* in the Rho*–GT* complex was determined by a [³⁵S]GTPγS binding assay and the extinction coefficient for the chromophore retinal in Rho*. (This experiment was repeated three times.) D, SEC profiles of the Rho*–GT* complex after 1 day (blue) and 7 days (orange). (This is representative of more than 20 similar sets of SEC profiles.)

Figure 4.

SAXS data and analyses of the Rho*–G_T* complex. A, SAXS scattering profiles from protein concentrations of 1, 0.5, and 0.25 mg/ml (colored cyan, purple, and red, respectively). (Each curve shown is the average of 10 scattering curves.) B, Guinier plots for q*R_g <1.3 region of the scattering curve. C, SAXS parameters and calculation results. D, pair distribution function calculated from 1 mg/ml scattering profile.

Figure 5.

Ab initio envelopes and model of the Rho*–G_T* complex. A, DAMMIN envelopes (averaged and filtered envelopes are colored in gray and blue, respectively). B, GASBOR envelope. C, superimposition of the Rho*–G_T* complex model with the GASBOR envelope. Rho* is in red; the α_T* GTPase domain is in green; the α_T* helical domain is in yellow; β₁ is in cyan; γ₁ is in gray; and detergents are shown as orange beads. D, theoretical scattering profile of the Rho*–G_T* complex model (red line) overlaid with experimental data (gray dots). E, position of the α_T* helical domain (yellow) in the model compared with open (purple) and closed (blue) positions from X-ray crystal structures.

Figure 6.

EM 2D projection analysis of the Rho*–G_T* complex. A, raw EM image of the detergent-solubilized Rho*–G_T* complex embedded in negative stain. B, representative EM class averages of the Rho*–G_T* complex (the positions of the α_Τ* helical domain are indicated by an arrow). The schematic model represents the conformations reflected by the EM averages, depicting the variable positioning of the helical domain (the position of the detergent micelle is indicated by gray shaded arcs and labeled with m).